The Study Of Protective Effect Of Astragalus Mongholicus Polysaccharides On LPS-induced Damage In Intestinal Epithelial Cell

ObjectiveIntestinal epithelial cell is an important component of intestinal mucosal barrier.It is the first mucosal surface of the body and considered as acquired immune systems of the host which plays a central role in regulating.It is also the first two-way connection defense between pathogenic microorganisms and the host.Some non-intestinal diseases and gastrointestinal diseases can cause intestinal dysfunction.When severe stress state, the systemic immune function is low.A large number of intestinal bacteria and endotoxin intruse circulation and cause intestinal bacterial translocation and endotoxemia,which will further aggravate the intestinal epithelial cell injury,activate a series of immune response,result in a number of inflammatory mediators such as TNF-α,IL-6,IL-8,platelet activating factor.A large number of production can cause systemic inflammatory response syndrome(SIRS),activate and accelerate the multi-system organ failure(MSOF).Research on LPS-mediated intestinal epithelial cell injury has become a widespread area of study.Much progress has been made in recent years.NF-κB is the key factor of the expression of many genes regulated the intestinal immune function,which play an indispensable role for many cytokines(IL-1,IL-2,IL-6,IL-8,IL-12) expressed in lymphocytes,epithelial cells and mononuclear cells.Now clear that,NF-κB in intestinal epithelial cells may be activated when stimulated by LPS.NF-κB can induce the increase in gene expression of multi-cytokine (IL-1,IL-6,IL-8,TNF),adhesion molecules,chemokines factor and the acute phase protein.Therefore,the NF-κB,as a transcription factor of the continue transformation in the gastrointestinal tract,regulate the expression of inflammatory genes,trigger and expand the inflammatory response,result in the occurrence of disease.Since the activation of NF-κB is known as an important pathway leading to intestinal epithelial cell injury,the study on blocking NF-κB activation in order to control and inhibit inflammatory response is a promising way in the treatment of gastro-intestinal injury.Mitogen-activated protein kinase(MAPK) is the serine/threonine residues of the protein kinase widely distributed within the cells.It is also an important signal regulatory enzymes connected the cell surface receptor with its decisive gene expression.In mammalian cells,it have been cloned at least four MAPK family: respectively extracellular signal- regulated kinase(ERK1/ERK2),cJun N-terminal kinase(JNK1/JNK 2),P38 MAPK(α,β) and ERK5.The activation of MAPK can stay in the cytoplasm to range other protein kinase.As a result,cytoskeletal components phosphorylate.MAPK is activated by phosphorylation,translocated to the nucleus, which trigger its transcription factor gene expression and promote the protein synthesis.Grishin and other scholars found that P38MAPK and COX-2 phosphorylation expression increased when IEC-6 cells stimulated by LPS in vitro.The phosphorylation of P38 induced by LPS in IEC-6 with a dose-dependent manner.The study had confirmed that P38MAPK involved in the inflammation of the intestinal epithelial cells induced by the LPS in vitro.MAPK affect gene expression,the media release and other cell signaling pathways through a multi-level,multi-faceted signal conditioning.It involved the release of pro-inflammatory cytokines and apoptosis injury in the intestinal cells.It can be believed that blocking and controlling MAPK signaling molecule expression and activity of intestinal injury will provide new ideas and approaches.Astragalus is our traditional Chinese medicine.Research shows that the Astragalus can promote normal development of immune organs and impaired immune organ recovery,enhance and restore immune function.It has been used to control tumor,immunocompromised and immunodeficiency diseases in China.Astragalus mongholicus polysaccharides(APS) is an important component of Astragalus.Many studies have confirmed that the APS regulate immune and stimulate the hematopoietic function.Wang Li xin found that APS antagonized the drug from the liver homogenate index of malondialdehyde(MDA) levels and reduced glutathione(GSH).Lu Jing Tao has confirmed the APS can inhibit lipopolysaccharide-induced rat peritoneal macrophages releasing of tumor necrosis factor,interleukin-1 and the secretion of nitric oxide.It showed that APS can inhibit the secretion of inflammatory cytokines thereby reducing tissue injury to play its role in immune protection.At home and abroad,no study was done on whether the APS has a protective effect in intestinal injury caused by LPS,as well as whether the protective effect of APS related with some signal transduction pathway.After the IEC-6 cells were stimulated by LPS,We studied whether the APS can effectively promote cell proliferation,whether APS plays the role in the protection of intestinal realated with the signal of MAPK/NF-κB transduction pathway.Through the study,we explore the mechanism of APS in intestinal protection so as to provide a theoretical basis for clinical application.Material and methods1.MaterialsIEC-6 cells were purchased from the Test Center of Chinese Academy of Medical Sciences.IEC-6 cells were cultured with DMEM medium supplemented with 10%fetal bovine serum and 0.01 mg/ml insulin.IEC-6 ceils were cultured in the CO₂ incubator(37℃,5%CO₂).It is soluted for the next day to about 80 per cent covered, then incubated with trypsin(0.25%).Cells from passage 5 were used for study.The cultured cells will be divided into 5 groups:Group 1:the control group,add DMEM only for control;Group 2:LPS group,the cultured cells by adding LPS(10 ug/ ml);Group 3:APS group:it divide into 4 sub-group:â‘ APS50 ug/ml;â‘¡APS100 ug/ml;â‘¢APS200 ug/ml;â‘£APSS00 ug/ml;Group 4:LPS and different concentration APS group:it divide into 4 sub-group:â‘ LPS10 ug/ml+APS50ug/ml;â‘¡LPS10 ug/ml +APS 100 ug/ml;â‘¢LPS10 ug/ml+APS 200 ug/ml;â‘£LPS10 ug/ml+APS 500 ug/ml; Group 5:APS and different concentration LPS group:it divide into 3 sub-group:â‘ APS500 ug/ml+LPS 5 ug/ml;â‘¡APS500 ug/ml+LPS 10 ug/ml;â‘¢APS500 ug/ml+LPS 20 ug/ml.2.MethodsThe rat small intestinal cell line IEC-6 was grown in Dulbecco's modified.Eagle's medium(DMEM) supplemented with 10%FBS and 0.01mg/ml insulin.IEC-6 cells were cultured at 37℃in a humidified atmosphere containing 5%CO2 with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of APS on normal IEC-6.The IEC-6 cells were stimulated with LPS(10ug/ml) for 1h,then added with various concentration of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.Cell proliferation was measured by MTT assay to observe the effects of APS on LPS- stimulated IEC-6.The IEC-6 cells were pretreated with APS(500ug/ml) for 24h,then added with various concentration of LPS(5ug/ml,10ug/ml,20ug/ml) for 1h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of pretreated APS on LPS-stimulated IEC-6.Reverse Transcriptase-Mediated PCR(RT-PCR) Analysis detect TNF-αmRNA and IL-8 mRNA.IEC-6 cells were culture in DMEM with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for24h,then added LPS(10ug/ml) for 1h and 4h to detect TNF-αmRNA and IL-8 mRNA.Western-Blotting assay to detect protein of P-ERK1/2,P-JNK,P-P38,NF-κB and IκB-α.The cells were cultured in DMEM medium with various concentrations of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,then added LPS for 15 min to detect IκB-α,for 30 min to detect NF-κB,and for 1h to detect phosphorylated-P38, phosphorylated-ERK1/2,phosphorylated-JNK.3.Statistical analysisSPSS11.5 was used to perform statistical analysis,with all data expressed as mean±SEM.Ststistically significantdifferences in the values were analyzed with ANOVA for inter-group comparion.P-value＜0.05 were considered statistically significant.Results1.Morphology change in IEC-6Normal intestinal epithelial cells were embedded as paving stones,not overlapping. A typical single-layer cell was seen clearly with larger nucleus.Intestinal epithelial cells stimulated by LPS were showed fuzzy boundary cells.A large number of cells membrane rupture and cell morphology was not integrity.2.The proliferation impact of APS on IEC-6 cells stimulated by LPS.APS can promote normal IEC-6 cell proliferation.MTT result showed:Different concentrations of APS showed a significant role in promoting IEC-6 cell proliferation(P＜0.05).APS on the IEC-6 cells has a dose-dependent role in promoting proliferation.With the extension of time,the effect of promoting cell proliferation gradually weakened.For the first 24- hour,the concentration of 50 ug/ ml APS can promote the cell proliferation significantly.However,in the 72- hour,the concentration of 200 ug/ ml APS displayed a remarkable effect of promoting cell proliferation.APS cannot promote IEC-6 cell proliferation which stimulated by LPS.MTT result showed:APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.Only in the first 24- hour,the concentration of 200 ug/ml and 500 ug/ml APS showed significant role in promoting cell proliferation.In the 48-hour and 72-hour,with the different concentrations of APS did not have effect of promoting cell proliferation(P＞0.05). Pretreated APS can promote IEC-6 cell proliferation which stimulated by LPS.MTT result showed:When IEC-6 cells were stimulated by the concentration of 5 ug/ml LPS,Pretreated APS do not promote cell proliferation on IEC-6 compared with the control group(P＞0.05).But when IEC-6 cells were stimulated by the concentration of 10 ug/ml and 20 ug/ml LPS,Pretreated APS group promote cell proliferation on IEC-6 compared with the control group(P＜0.01).The results indicate that, pretreatment with APS can reduce IEC-6 cell injury induced by LPS.3.APS inhibited the secretion of TNF-α,IL-8 mRNA production in IEC-6 cells stimulated by LPS.APS abrogates LPS-induced TNF-αmRNA expression in IEC-6.We first evaluted the effects of APS on LPS-induced TNF-αgene expression in intestinal cell line IEC-6.Stimulation of IEC-6 by LPS markedly increased the production of TNF-α.We examined the effect of APS on the mRNA level of TNF-αfrom IEC-6 when stimulated with LPS.Cells were pretreated with various concentrations of APS(50ug/ml,100ug/ml,200ug/ml,500ug/ml) for 24h,stimulated with LPS(10ug/ml)for 1h and TNF-αgene expression was measured by RT-PCR assay.RT-PCR analysis showed that TNF-αmRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner:50ug/ml of APS partially supressed TNF-αmRNA from LPS activated IEC-6,and 500ug/ml of APS significantly inhibited the amount of TNF-αmRNA.(P＜0.01 compared with LPS stimulation in the absence of APS).APS abrogates LPS-induced IL-8 mRNA expression in IEC-6.We then examined the effect of APS in the production of IL-8 from IEC-6 by LPS.IL-8 mRNA was induced readily from IEC-6 by LPS.However,the mRNA level of IL-8 was inhibited by APS in a concentration- dependent manner:addition of APS partially supressed IL-8 level at 50ug/ml and 100ug/ml,respectively,and 500ug/ml of APS significantly inhibited IL-8 at mRNA level.(P＜0.01 compared with LPS stimulation in the absence of APS). APS Inhibited both TNF-αand IL-8 Production from LPS-Activated IEC-6 in a time-dependent manner.Next,IEC-6 cells were pretreated with APS(500ug/ml) for 24h,stimulated with LPS(10ug/ml) for 1h to 4h and TNF-α,IL-8 gene expression was measured by RT-PCR.LPS-induced TNF-αmRNA expression is inhibited by APS treatment(10.3% and 25.5%inhibition at 1h and 4h poststimulation,respectively).LPS-induced IL-8 mRNA expression is inhibited by APS treatment(15.3%and 18.8%inhibition at 1h and 4h poststimulation,respectively).4.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.It has been reported that APS abrogates LPS-induced pro-inflammatory cytokine production in IEC-6.A critical downstream effector pathway induced by LPS is the NF-κB transcriptional system.NF-κB is usually held in an inactivated state in the cytoplasm of unstimulated cells through interaction with IκB-α(NF-κB inhibitory protein).After stimulation with LPS,the IκB-αis degraded and release the NF-κB to translocate into nucleus,where NF-κB drive the expression of pro-inflammatory cytokines.Thus we next assessed whether APS abrogates LPS-induced NF-κB transcriptional activity.To do this,we examined whether APS blocks NF-κB translocation into nucleus,since nuclear translocation is required for NF-κB-Mediated transcriptional activation.We examined the levels of NF-κB in the nucleus of LPS-stimulated IEC-6 cells in the presence or absence of APS.LPS stimulation resulted in an increase in protein level in the nucleus.Treatment with APS abolished this effect.To dissect the effect of APS on LPS-induced NF-κB signal,we next determined IκB-αprotein involved in NF-κB pathway using Western blot analysis.It shows that LPS decrease IκB-αin IEC-6cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.5.The analysis of the expression of MAPK protein in IEC-6 cells induced by APS.It is reported that the activation of the MAPKS P38 is important signaling pathways responsible for the pro-duction of pro-inflammatory cytokines when IEC-6 is activated.In order to further understand the mechanisms of APS-mediated anti-inflammation in IEC-6,we examined whether APS inhibits LPS-triggered activation of MAPK signalling incluing P38,ERK1/2 and JNK.The active phosphorylated levels of P38,ERK1/2 and JNK were analyzed in LPS-stimulated IEC-6 cells following treatment with or without APS.Results indicated that P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.P38,ERK1/2 and JNK were all activated in IEC-6 in lh after LPS treatment.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.When 50ug/ml of APS is added,LPS-induced P38 was partially blocked in IEC-6,and 500ug/ml of APS significantly inhibited P38 phosphorylation in LPS-stimulated IEC-6.However,APS did not alter the activation of the protein levels of ERK1/2 and JNK.The observations indicate that APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.Conclusions1.APS has a role of promoting the normal IEC-6 cell proliferation.APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.When IEC-6 cells were pretreated with APS,APS can promote cell proliferation compared with the control group.The results indicate that pretreatment with APS can reduce IEC-6 cell injury induced by LPS.2.APS abrogates LPS-induced TNF-αand IL-8 mRNA expression in IEC-6. TNF-αand IL-8 mRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner.We also confirm APS innhibited both TNF-αand IL-8 production from LPS-Activated IEC-6 in a time-dependent manner.3.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.LPS stimulation resulted in an increase in NF-κB protein level in the nucleus.Treatment with APS abolished this effect.LPS decrease IκB-αin IEC-6 cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.4.P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.APS did not alter the activation of the protein levels of ERK1/2 and JNK phosphorylation.APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.